Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Modifications have been identified that can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein's stability, circulation time, and biological activity. A review article describing protein modification and fusion proteins is Francis (1992), Focus on Growth Factors 3:4-10 (Mediscript, London), which is hereby incorporated by reference.
One useful modification is combination of a polypeptide with an “Fc” domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab,” which binds antigen, and a constant domain known as “Fc,” which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337: 525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.
TABLE 1Fc fusion with therapeutic proteinsFusionTherapeuticForm of FcpartnerimplicationsReferenceIgG1N-terminus ofHodgkin's disease;U.S. Pat. No. 5,480,981CD30-Lanaplastic lymphoma;T-cell leukemiaMurine Fcγ2aIL-10anti-inflammatory;Zheng et al. (1995), J.transplant rejectionImmunol. 154:5590-600IgG1TNF receptorseptic shockFisher et al. (1996), N.Engl. J. Med. 334: 1697-1702; Van Zee, K.et al. (1996). J.Immunol. 156: 2221-30IgG, IgA, IgM,TNF receptorinflammation, autoimmuneU.S. Pat. No. 5,808,029,or IgEdisordersissued Sep. 15, 1998(excluding thefirst domain)IgG1CD4 receptorAIDSCapon et al. (1989), Nature337: 525-31IgG1,N-terminusanti-cancer, antiviralHarvill et al. (1995),IgG3of IL-2Immunotech. 1: 95-105IgG1C-terminus ofosteoarthritis;WO 97/23614, publishedOPGbone densityJul. 3, 1997IgG1N-terminus ofanti-obesityPCT/US 97/23183, filedleptinDec. 11, 1997Human IgCTLA-4autoimmune disordersLinsley (1991), J. Exp.C□1Med. 174: 561-9
Polyethylene glycol (“PEG”) fusion proteins and peptides have also been studied for use in pharmaceuticals, on artificial implants, and other applications where biocompatibility is of importance. Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to pharmaceuticals and implants and to molecules and surfaces generally. For example, PEG derivatives have been proposed for coupling PEG to surfaces to control wetting, static buildup, and attachment of other types of molecules to the surface, including proteins or protein residues.
In other studies, coupling of PEG (“PEGylation”) has been shown to be desirable in overcoming obstacles encountered in clinical use of biologically active molecules. Published PCT Publication No. WO 92/16221 states, for example, that many potentially therapeutic proteins have been found to have a short half life in blood serum.
PEGylation decreases the rate of clearance from the bloodstream by increasing the apparent molecular weight of the molecule. Up to a certain size, the rate of glomerular filtration of proteins is inversely proportional to the size of the protein. The ability of PEGylation to decrease clearance, therefore, is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Decreased clearance can lead to increased efficacy over the non-PEGylated material. See, for example, Conforti et al., Pharm. Research Commun. vol. 19, pg. 287 (1987) and Katre et., Proc. Natl. Acad. Sci. U.S.A. vol. 84, pg. 1487 (1987).
In addition, PEGylation can decrease protein aggregation, (Suzuki et al., Biochem. Biophys. Acta vol. 788, pg. 248 (1984)), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. vol. 252 pg. 3582 (1977)), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221.
PEGylation of proteins illustrates some of the problems that have been encountered in attaching PEG to surfaces and molecules. The vast majority of PEGylating reagents react with free primary amino groups of the polypeptide. Most of these free amines are the epsilon amino group of lysine amino acid residues. Typical proteins possess a large number of lysines. Consequently, random attachment of multiple PEG molecules often occurs leading to loss of protein activity.
In addition, if the PEGylated protein is intended for therapeutic use, the multiple species mixture that results from the use of non-specific PEGylation leads to difficulties in the preparation of a product with reproducible and characterizable properties. This non-specific PEGylation makes it difficult to evaluate therapeutics and to establish efficacy and dosing information. Site selective PEGylation of such proteins, however, leads to reproducibly-modified materials that gain the desirable attributes of PEGylation without the loss of activity.
A variety of means have been used to attach the polyethylene glycol molecules to the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. For example, Royer (U.S. Pat. No. 4,002,531, incorporated herein by reference in its entirety) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. EP 0 539 167 (incorporated herein by reference in its entirety), published Apr. 28, 1993, Wright, “Peg Imidates and Protein Derivates Thereof” states that peptides and organic compounds with free amino group(s) are modified with a derivative of PEG or related water-soluble organic polymers. U.S. Pat. No. 4,904,584 (incorporated herein by reference in its entirety), Shaw, issued Feb. 27, 1990, relates to the modification of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. An increasing number of therapeutic proteins modified in the manner described above to include PEG have been previously described. See also, for example, European patent application publications EP 0 401 384; EP 0 473 268; EP 0 335 423; EP 0 442 724; EP 0 154 316, each of which is incorporated by reference in its entirety.
Other methods for attaching a polymer to a protein involve using a moiety to act as a linking group. Such moieties may, however, be antigenic. A tresyl chloride method involving no linking group is available, but this method may be difficult to use to produce therapeutic products as the use of tresyl chloride may produce toxic by-products. See Francis et al., In: Stability of protein pharmaceuticals: in vivo pathways of degradation and strategies for protein stabilization (Eds. Ahern., T. and Manning, M. C.) Plenum, N.Y., 1991) and Delgado et al., “Coupling of PEG to Protein By Activation With Tresyl Chloride, Applications In Immunoaffinity Cell Preparation”, In: Fisher et al., eds., Separations Using Aqueous Phase Systems, Applications In Cell Biology and Biotechnology, Plenum Press, N.Y. N.Y., 1989 pp. 211-213, each of which is incorporated by reference in its entirety.
In general, the interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated in the case of human growth hormone bound to its receptor, only a few key residues at the interface actually contribute to most of the binding energy. Clackson, T. et al., Science 267:383-386 (1995). This observation and the fact that the bulk of the remaining protein ligand serves only to display the binding epitopes in the right topology makes it possible to find active ligands of much smaller size. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).
Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998, each of which is incorporated by reference. In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation, and the best binding peptides are sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.
Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). These and related methods are collectively referred to as “E. coli display.” Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion. See Smith et al. (1993), Mol. Pharmacol. 43: 741-8. The method of Smith et al. and related methods are referred to as “yeast-based screening.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. This and related methods are collectively referred to as “ribosome display.” Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. This and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. These and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.
In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be carried out. In such an approach, stepwise changes are made to a peptide sequence and the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure) is determined. These techniques are collectively referred to as “rational design.” In one such technique, a series of peptides is made in which a single residue at a time is replaced with alanine. This technique is commonly referred to as an “alanine walk” or an “alanine scan.” When two residues (contiguous or spaced apart) are replaced, it is referred to as a “double alanine walk.” The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties.
Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These and related methods are referred to as “protein structural analysis.” These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.
Conceptually, peptide mimetics of any protein can be identified using phage display and the other methods mentioned above. These methods have also been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin. Biotech. 7: 616-21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19-20.
Of particular interest is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefor in parentheses. Some of these peptides have been modified (e.g., to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein (e.g., EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monoclonal antibody.
TABLE 2Pharmacologically active peptidesBindingpartner/Form ofprotein ofPharmacologicpeptideinterest1activityReferenceintrapeptideEPO receptorEPO-mimeticWrighton et al. (1996),disulfide-Science 273: 458-63; U.S.bondedPat. No. 5,773,569, issuedJun. 30, 1998 to Wrightonet al.C-terminallyEPO receptorEPO-mimeticLivnah et al. (1996),cross-linkedScience 273: 464-71;dimerWrighton et al. (1997),Nature Biotechnology 15:1261-5; International patentapplication WO 96/40772,published Dec. 19, 1996linearEPO receptorEPO-mimeticNaranda et al. (1999), Proc.Natl. Acad. Sci. USA, 96:7569-74; WO 99/47151,published Sep. 23, 1999linearc-MplTPO-mimeticCwirla et al.(1997) Science276: 1696-9; U.S. Pat. No.5,869,451, issued Feb. 9,1999; U.S. Pat. No.5,932,946, issued Aug. 3,1999C-terminallyc-MplTPO-mimeticCwirla et al. (1997),cross-linkedScience 276: 1696-9dimerdisulfide-stimulation of hematopoiesisPaukovits et al. (1984),linked dimer(“G-CSF-mimetic”)Hoppe-Seylers Z. Physiol.Chem. 365: 303-11;Laerum et al. (1988), Exp.Hemat. 16: 274-80alkylene-G-CSF-mimeticBhatnagar et al. (1996),linked dimerJ. Med. Chem. 39: 3814-9;Cuthbertson et al. (1997), J.Med. Chem. 40: 2876-82;King et al. (1991), Exp.Hematol. 19: 481; King etal. (1995), Blood 86(Suppl. 1): 309alinearIL-1 receptorinflammatory andU.S. Pat. No. 5,608,035;autoimmune diseasesU.S. Pat. No. 5,786,331;(“IL-1 antagonist” orU.S. Pat. No. 5,880,096;“IL-1ra-mimetic”)Yanofsky et al. (1996).Proc. Natl. Acad. Sci. 93:7381-6; Akeson et al.(1996), J. Biol. Chem. 271:30517-23; Wiekzorek et al.(1997), Pol. J. Pharmacol.49: 107-17; Yanofsky(1996), PNAs, 93: 7381-7386.linearFacteur thymiquestimulation of lymphocytesInagaki-Ohara et al. (1996),serique (FTS)(“FTS-mimetic”)Cellular Immunol. 171: 30-40; Yoshida (1984), Int. J.Immunopharmacol, 6: 141-6.intrapeptideCTLA4 MAbCTLA4-mimeticFukumoto et al. (1998),disulfideNature Biotech. 16: 267-70bondedexocyclicTNF-α receptorTNF-α antagonistTakasaki et al. (1997),Nature Biotech. 15: 1266-70; WO 98/53842,published Dec. 3, 1998linearTNF-α receptorTNF-α antagonistChirinos-Rojas ( ), J.Imm., 5621-5626.intrapeptideC3binhibition of complementSahu et al. (1996), J.disulfideactivation; autoimmuneImmunol. 157: 884-91;bondeddiseasesMorikis et al. (1998),(“C3b-antagonist”)Protein Sci. 7: 619-27linearvinculincell adhesion processes-Adey et al. (1997).cell growth, differentiation,Biochem. J. 324: 523-8wound healing, tumormetastasis (“vinculinbinding”)linearC4 bindinganti-thromboticLinse et al. (1997), J. Biol.protein (C4BP)Chem. 272: 14658-65linearurokinase receptorprocesses associated withGoodson et al. (1994),urokinase interaction with itsProc. Natl. Acad. Sci. 91:receptor (e.g., angiogenesis,7129-33; Internationaltumor cell invasion andapplication WO 97/35969,metastasis); (“UKRpublished Oct. 2, 1997antagonist”)linearMdm2, Hdm2Inhibition of inactivation ofPicksley et al. (1994),p53 mediated by Mdm2 orOncogene 9: 2523-9;hdm2; anti-tumorBottger et al. (1997) J. Mol.(“Mdm/hdm antagonist”)Biol. 269: 744-56; Bottgeret al. (1996), Oncogene 13:2141-7linearp21WAF1anti-tumor by mimicking theBall et al. (1997), Curr.activity of p21WAF1Biol. 7: 71-80linearfarnesylanti-cancer by preventingGibbs et al. (1994), Celltransferaseactivation of ras oncogene77: 175-178linearRas effectoranti-cancer by inhibitingMoodie et al. (1994),domainbiological function of the rasTrends Genet 10: 44-48oncogeneRodriguez et al. (1994),Nature 370: 527-532linearSH2/SH3anti-cancer by inhibitingPawson et al (1993), Curr.domainstumor growth with activatedBiol. 3: 434-432tyrosine kinases; treatmentYu et al. (1994), Cellof SH3-mediated disease76: 933-945; Rickles et al.states (“SH3 antagonist”)(1994), EMBO J. 13: 5598-5604; Sparks et al. (1994),J. Biol. Chem. 269: 23853-6; Sparks et al. (1996),Proc. Natl. Acad. Sci. 93:1540-4; U.S. Pat. No.5,886,150, issued Mar.23, 1999; U.S. Pat. No.5,888,763, issued Mar.30, 1999linearp16INK4anti-cancer by mimickingFåhraeus et al. (1996),activity of p16; e.g.,Curr. Biol. 6: 84-91inhibiting cyclin D-Cdkcomplex (“p16-mimetic”)linearSrc, Lyninhibition of Mast cellStauffer et al. (1997),activation, IgE-relatedBiochem. 36: 9388-94conditions, type Ihypersensitivity (“Mast cellantagonist”)linearMast cell proteasetreatment of inflammatoryInternational applicationdisorders mediated byWO 98/33812, publishedrelease of tryptase-6Aug. 6, 1998(“Mast cell proteaseinhibitors”)linearHBV core antigentreatment of HBV viralDyson & Muray (1995),(HBcAg)infections (“anti-HBV”)Proc. Natl. Acad. Sci. 92:2194-8linearselectinsneutrophil adhesion;Martens et al. (1995), J.inflammatory diseasesBiol. Chem. 270: 21129-(“selectin antagonist”)36; European patentapplication EP 0 714 912,published Jun. 5, 1996linear, cyclizedcalmodulincalmodulin antagonistPierce et al. (1995), Molec.Diversity 1: 259-65;Dedman et al. (1993), J.Biol. Chem. 268: 23025-30; Adey & Kay (1996),Gene 169: 133-4linear,integrinstumor-homing; treatment forInternational applicationscyclized-conditions related toWO 95/14714, publishedintegrin-mediated cellularJun. 1, 1995; WOevents, including platelet97/08203, published Mar.aggregation, thrombosis,6, 1997; WO 98/10795,wound healing,published Mar. 19, 1998;osteoporosis, tissue repair,WO 99/24462, publishedangiogenesis (e.g., forMay 20, 1999; Kraft et al.treatment of cancer), and(1999), J. Biol. Chem. 274:tumor invasion1979-1985(“integrin-binding”)cyclic, linearfibronectin andtreatment of inflammatoryWO 98/09985, publishedextracellularand autoimmune conditionsMar. 12, 1998matrixcomponents of Tcells andmacrophageslinearsomatostatin andtreatment or prevention ofEuropean patent applicationcortistatinhormone-producing tumors,0 911 393, published Apr.acromegaly, giantism,28, 1999dementia, gastric ulcer,tumor growth, inhibition ofhormone secretion,modulation of sleep orneural activitylinearbacterialantibiotic; septic shock;U.S. Pat. No. 5,877,151,lipopolysaccharidedisorders modulatable byissued Mar. 2, 1999CAP37linear orpardaxin, mellitinantipathogenicWO 97/31019, publishedcyclic,28 Aug. 1997including D-amino acidslinear, cyclicVIPimpotence,WO 97/40070, publishedneurodegenerative disordersOct. 30, 1997linearCTLscancerEP 0 770 624, publishedMay 2, 1997linearTHF-gamma2Burnstein (1988),Biochem., 27: 4066-71.linearAmylinCooper (1987), Proc. Natl.Acad. Sci., 84: 8628-32.linearAdrenomedullinKitamura (1993), BBRC,192: 553-60.cyclic, linearVEGFanti-angiogenic; cancer,Fairbrother (1998),rheumatoid arthritis, diabeticBiochem., 37: 17754-17764.retinopathy, psoriasis(“VEGF antagonist”)cyclicMMPinflammation andKoivunen (1999), Natureautoimmune disorders;Biotech., 17: 768-774.tumor growth(“MMP inhibitor”)HGH fragmenttreatment of obesityU.S. Pat. No. 5,869,452Echistatininhibition of plateletGan (1988). J. Biol. Chem.,aggregation263: 19827-32.linearSLE autoantibodySLEWO 96/30057, publishedOct. 3, 1996GD1alphasuppression of tumorIshikawa et al. (1998),metastasisFEBS Lett. 441 (1): 20-4antiphospholipidendothelial cell activation,Blank et al. (1999), Proc.beta-2-antiphospholipid syndromeNatl. Acad. Sci. USA 96:glycoprotein-I(APS), thromboembolic5164-8(β2GPI)phenomena,antibodiesthrombocytopenia, andrecurrent fetal losslinearT Cell ReceptordiabetesWO 96/11214, publishedbeta chainApr. 18, 1996.Antiproliferative, antiviralWO 00/01402, publishedJan. 13, 2000.anti-ischemic, growthWO 99/62539, publishedhormone-liberatingDec. 9, 1999.anti-angiogenicWO 99/61476, publishedDec. 2, 1999.linearApoptosis agonist; treatmentWO 99/38526, publishedof T cell-associatedAug. 5, 1999.disorders (e.g., autoimmunediseases, viral infection, Tcell leukemia, T celllymphoma)linearMHC class IItreatment of autoimmuneU.S. Pat. No. 5,880,103,diseasesissued Mar. 9, 1999.linearandrogen R, p75,proapoptotic, useful inWO 99/45944, publishedMJD, DCC,treating cancerSep. 16, 1999.huntingtinlinearvon Willebrandinhibition of Factor VIIIWO 97/41220, publishedFactor; Factor VIIIinteraction; anticoagulantsApr. 29, 1997,linearlentivirus LLP1antimicrobialU.S. Pat. No. 5,945,507,issued Aug. 31, 1999.linearDelta-Sleepsleep disordersGraf (1986), PeptidesInducing Peptide7: 1165.linearC-Reactiveinflammation and cancerBarna (1994), CancerProtein (CRP)Immunol. Immunother.38: 38 (1994).linearSperm-ActivatinginfertilitySuzuki (1992), Comp.PeptidesBiochem. Physiol.102B: 679.linearangiotensinshematopoietic factors forLundergan (1999), J.hematocytopenic conditionsPeriodontal Res. 34(4):from cancer, AIDS, etc.223-228.linearHIV-1 gp41anti-AIDSChan (1998), Cell 93:681-684.linearPKCinhibition of bone resorptionMoonga (1998), Exp.Physiol. 83: 717-725.lineardefensins (HNP-antimicrobialHarvig (1994), Methods1, -2, -3, -4)Enz. 236: 160-172.linearp185HER2/neu,AHNP-mimetic: anti-tumorPark (2000), Nat.C-erbB-2Biotechnol. 18: 194-198.lineargp130IL-6 antagonistWO 99/60013, publishedNov. 25, 1999.linearcollagen, otherautoimmune diseasesWO 99/50282, publishedjoint, cartilage,Oct. 7, 1999.arthritis-relatedproteinslinearHIV-1 envelopetreatment of neurologicalWO 99/51254, publishedproteindegenerative diseasesOct. 14, 1999.linearIL-2autoimmune disorders (e.g.,WO 00/04048, publishedgraft rejection, rheumatoidJan. 27, 2000; WOarthritis)00/11028, published Mar.2, 2000.1The protein listed in this column may be bound by the associated peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.
Peptides identified by peptide library screening have been regarded as “leads” in development of therapeutic agents rather than being used as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, the art presently uses the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8.
In spite of the availability of recombinant therapeutic proteins, previously identified peptide mimetic, and modifications thereof, there thus remains a need in the art to provide additional compounds that have improved bioefficacy, particularly when administered in a multidose regimen.